Extracorporal system for treating disease with radionucleotides

ABSTRACT

A composition of matter comprises a pathogen-targeting organic moiety which is conjugated to a radioisotope which has a half-life of less than 100 days. The composition can be synthesized by bringing together a radioisotope having a half life of less than 100 days with a greater than stoichiometric amount of a complexing agent so as to form a first mixture containing a reaction product between the radioisotope and the complexing agent; removing the excess complexing agent from the mixture; and bringing together the first reaction product and an antibody substance so as to form a second mixture containing a reaction product between the first reaction product and the antibody substance. The composition is deposited on a particulate substrate and is useful for treating infectious diseases caused by pathogens by passing the patients blood through a bed formed from the particles.

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of copendingapplication Ser. No. 09/183,454, filed Oct. 30, 1998, now , andincorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] This invention relates to the delivery of radioisotopes to adisease-causing pathogen using a pathogen-targeting material conjugatedto the radioisotope.

[0003] It is known to deliver cytotoxic radioisotopes to the nucleus ofa tumor cell using a targeting protein or polypeptide conjugated with aradio-labeled nucleic acid-targeting small molecule. See, for example,U.S. Pat. No. 5,759,514. However, other than in the above referencedapplication, the use of radioisotopes to destroy disease-causing livingpathogens such as bacteria or viruses has not heretofore been suggested.

[0004] Some strains of bacteria and viruses are very resistant toconventional drug therapy and are capable of killing or seriouslydebilitating the patient. Some strains are capable of mutating into apredominantly drug resistant form during the course of drug treatment,resulting in the death or debilitation of the patient. The widespreaduse of a particular drug treatment furthermore favors the geneticselection of strains which are resistant to that particular course oftreatment. The presence of drug resistant strains of bacteria andviruses poses a growing world wide health threat.

[0005] A method for treating patients which have been infected with adrug-resistant pathogen would be very desirable. A technique forperforming such treatment in a manner that minimizes the degree to whichpatients are exposed to a radioisotope would be even more desirable.

SUMMARY OF THE INVENTION

[0006] One embodiment of the invention provides a composition of mattersuitable for use to treat disease in an extracorporal treatmenttechnique. The composition comprises a conjugate of a livingpathogen-targeting organic moiety coupled to a radioisotope which has ahalf-life of less than 100 days on a suitable support.

[0007] Another embodiment of the invention provides a method fortreating an infectious disease caused by living blood-borne pathogens ina mammal. The process is conducted by obtaining antibodies from themammal, replicating the antibodies to produce replicated antibodies,conjugating the replicated antibodies with a radioisotope which has ahalf-life of less than 100 days to produce a conjugate, fixing theconjugate to a conjugate support to form a supported conjugate, andpassing the blood of the mammal into contact with the supportedconjugate to bring the conjugate into contact with said livingpathogens.

[0008] A further embodiment of the invention provides a method fortreating an infectious disease caused by living blood-borne pathogens ina mammal. The method is carried out by identifying the blood-bornepathogens causing the infectious disease, selecting a supportedconjugate comprising a particle support bearing an organic moiety whichis chemically selective for attachment to said living pathogens andwhich is conjugated to a radioisotope which has a half-life of less than100 days, flowing the blood of the mammal through a bed formed fromparticles of said supported conjugate, so that said blood-bornepathogens become associated with said radioisotope while in the bed,forming treated blood, and returning the treated blood to the mammal.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] In one embodiment of the invention, there is provided acomposition of matter comprising a living pathogen-targeting organicmoiety which is conjugated to a radioisotope which has a half-life ofless than 100 days and is deposited on a particulate substrate such asbeads to facilitate an extracorporal treatment of the patient's bodyfluid, such as blood.

[0010] Recent evidence has shown that radioisotopes which emit alpha,beta, or gamma radiation, and especially those of fairly short half-lifeand which emit Auger electrons during the decay process may be usefulfor inducing receptor cell specific cytotoxicity.

[0011] When a radioisotope decays by orbital electron capture orinternal conversion, inner atomic shell vacancies are created in theresidual atom. This highly excited atom attains a stable electronicconfiguration rapidly in a time scale of about 10⁻¹⁵ seconds viaradioactive and non-radioactive transitions. In general, Auger,Coster-Kronig and super Coster-Kronig processes dominate the atomicvacancy cascades. As a result, numerous electrons are ejected from theatom and most of these Auger electrons have very low kinetic energies(about 20-500 eV) with extremely short ranges (a few nanometers) inwater. Even though the energy carried by each of these electrons is onlya small fraction of the total energy released in the decay process,their collective energy deposition is extremely high. Hence when thedecays occur in the immediate vicinity of the critical biologicalmolecules such as DNA, intracellular transmitters or any of theapoptotic cascade mechanisms, the biological effects to that cell areexpected to be devastating.

[0012] Usually, radioisotopes used in accordance with the invention willhave a half-life in the range of from about 1 to about 10 days.Preferably, the radioisotopes emit Auger electrons. Examples of suitableradioisotopes are Phosphorus 32, Copper 67, Gallium 67, Bromine 77,Yttrium 90, Technetium 99, Indium 111, Iodine 125, Iodine 131, Rhenium186, Rhenium 188, Platinum 195, Bismuth 213, and Astatine 225. Of these,Copper 67, Yttrium 90, Indium 111, Rhenium 186, and Platinum 195 arepreferred because these radioisotopes have distinct cytotoxic propertieswhich may be exploited for therapy by the biologically directedtargeting.

[0013] Compounds that are labeled with Auger electron emitters are mosteffective when the compound is internalized within or attached to thecell in a manner capable of activating apoptosis. Auger electronsprovide very high-energy emissions but do so over a very short distanceor action, which is less than 10-20 microns. This allows for an Augeremitting radioisotope to bring a high energy destructive force intoareas to cause critical DNA strand damage (mitochondrial or nuclear).This, in turn activates the mechanism of apoptosis. Therefore, for aradioisotope-ligand to be a particularly desirable therapeutic agent,the compound must have a high cell to be destroyed-to background tissueratio, a high therapeutic ratio and pharmacokinetic biodistributionprofiles that optimize receptor binding, ligand internalization andcellular retention. The effects, therefore, of Auger electron emittersdepend upon their cellular and sub-cellular location, which is governed,in turn, by the chemical form of the molecular agent (bioactivesubstance) to which the radioisotope has been attached.

[0014] Generally speaking, the living pathogen-targeting organic moietyis in the form of an antiviral, antifungal or an antibacterial antibody,although fragments of such antibodies or antibiotics which function toselectively carry the radioisotope into or onto a targeted pathogen arealso considered suitable. Viruses, fungi bacteria, or prions may beselected as targets by appropriate selection of the organic moiety.Usually, the organic moiety has a surface chemistry which associateswith a surface chemistry of the targeted pathogen. More preferably, theorganic moiety has a surface chemistry to associate with a uniquesurface chemistry of the targeted pathogen.

[0015] Circulating antibodies normally recognize an antigen in the serumor tissue fluids and, furthermore, there are five identifiable classes:IgG, IgA, IgM, IgD and IgE. In addition to antigen binding, allantibodies exert other specific biological activities. Theantigen-binding site is usually one in which there is a Fc fragment andtwo-antigen binding FAB fragments. X-ray crystallography and electronmicroscopy has provided the structural and biochemical organization ofthese moieties. Disulfide bonds predominate in cross-linking many ofthese domains. The primary function of any antibody is to bind anyrecognizable antigen. Recently, libraries of human specific antibodyvariable genes have been constructed for recombinant filamentous phages,which display the antibodies on their surface, and it is possible toselect from high affinity antibodies for any chosen cell surfaceantigens from these libraries

[0016] Phage antibodies that bind to a particular antigen may beseparated from non-binding phage antibodies by antigen selection and thebound antibodies are recovered by elution. Repeated rounds of selectioncan isolate antigen-binding phages that were present at the start of theprocess at frequencies of less than one in a billion.

[0017] One technique of producing a homologous population of antibodiesof known antigen specificity, are known as hybridomas that are derivedfrom a single B cells and are called monoclonal antibodies. Anothertechnique for producing antibody molecules is named phage antibody orphage libraries. In this case, gene segments encoding antigen-bindingvariable or V domains of antibodies are fused to genes encoding the coatprotein of a bacteriophage. A collection of recombinant phage, eachdisplaying a different antigen-binding domain on its surface is known asa phage display library. Each phage isolated in this way win produce amonoclonal antigen-binding particle analogous to a monoclonal antibody.Genes encoding the antigen-binding site, which are unique to each phage,can then be recovered from the phage DNA and used to construct genes fora complete antibody molecule by joining them to gene segments thatencode the invariant parts of an antibody. When these reconstructedantibody genes are introduced into a suitable host cell line, thetransferred cells secrete antibodies with all of the desirablecharacteristics on monoclonal antibodies that are produced fromhybridomas.

[0018] The antibody binds stably to its antigen as the antibodiesrecognize the surface features of the native folded protein antigen andthe antibody molecules can thus be used to locate their target moleculesaccurately in single cells or in tissue sections.

[0019] Specific examples of bioactive substances that can be used asvectors for the radioisotopes include:

[0020] Biologicals:

[0021] A. Antiviral antibodies

[0022] 1. gp120 and gp41 for HIV virus,

[0023] 2. Anti elf-2 antibodies for vaccinia,

[0024] 3. Anti-gE/gl antibodies for herpes simplex 1 virus,

[0025] 4. Anti IL-10, or BHRF1 antibodies against the Ebstein-Barrvirus,

[0026] 5. Anti E3 antibodies for adenovirus group.

[0027] B. Antibacterial antibodies

[0028] 1. C3b, iC3b, MBL, certain oliosaaccharides, and lectins areidentifiable markers on gram positive and gram negative cell surfacesand thus serve as markers for vector antibodies.

[0029] C. Antibodies toward a unique family of proteins called STATsthat also bind DNA.

[0030] D. Antibodies directed toward surface LMP-1 antigens that arefound on the Ebstein-Barr infected cells, including the EB relatedpapillomas.

[0031] E. Antibiotics: Rimafin, streptomycin, tetracycline, puromycinand cyclohemimide all attach to RNA initiation, transcription mechanismof the rapidly reproducing bacteria but Actinomycin D which bindsdirectly to nuclear and mitochondrial DNA while the penicillins andcephalosporins bind directly to the chemokines of the cell wall of theinfecting organism. Radioisotopes that are conjugated to an antibioticwould not only enhance the bactericidal but bacteriostatic properties ofthe drug and but would also reduce the change of drug resistantbacteria.

[0032] F. Nonspecific apoptotic antibodies to: Fas ligand, IVCE/CED-3family, Bcl-2 family, NfkB, CD40L, CLTA-4, TNFR, APO, and TRAF familyactivate non-selectively the cell destructive mechanism or apoptosis

[0033] Labeling of biologics with radioisotopes for diagnosis andtherapy has usually been accomplished through the use of bifunctionalchelating agents, which contain both a reactive functionality forcovalent attachment to proteins and with a strong nucleotide bindinggroup capable of forming a chemically stable complex with theradioisotope. In the invention, a living pathogen-targeting organicmoiety is preferably covalently linked to a complexing agent which bindsthe radioisotope and the desired product from this coupling is depositedon a substrate. A preferred embodiment of the invention uses abifunctional complexing agent having a reactive functionality covalentlyattached to the pathogen-targeting organic moiety and a strongnucleotide binding group forming a chemically stable complex with theradioisotope. The strong nucleotide binding group can be in the form ofa chelating agent. Examples of suitable chelating agents which can beemployed in the invention include ethylenediaminetetraacetic acid(EDTA), diethylenetriaminopentaacetic acid (DTPA),ethyleneglycol-0,0′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA),N,N′-bis(hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED),triethylenetetraminehexaacetic acid (TTHA),1,4,7,10-tetra-azacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA),1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetraacetic acid (TITRA),1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA),1,4,8,11-tetraazacyclotetradecane (TETRA) and their substitutedderivatives. Of these, diethylenetriaminopentaacetic acid (DTPA) and1,4,7,10-tetra-azacyclododecane- N,N′,N″,N′″-tetraacetic acid (DOTA) arepreferred, because a method that we have modified and employedextensively for the radioactive labeling of bioactive molecules usesdiethylenetriaminopentaacetic acid (DTPA) and DOTA as bifunctionalchelates which may be achieved by using anhydride (mixed or cyclic) asacylating agents.

[0034] A preferred embodiment of the composition of matter can besynthesized by bringing together a radioisotope having a half life ofless than 100 days with a greater than stoichiometric amount of acomplexing agent so as to form a first mixture containing a reactionproduct between the radioisotope and the complexing agent; removing theexcess complexing agent from the mixture; and bringing together thefirst reaction product and an antibody substance so as to form a secondmixture containing a reaction product between the first reaction productand the antibody substance. Usually, the antibody substance comprises aprotein and generally will comprise an immunoglobulin produced bymammalian cells in response to a living pathogen. The complexing agenthas a first functional group which reacts with the radioisotope and asecond functional group which reacts with a site on the immunoglobulin.Preferably, the first reaction product is ionically neutral, and theprocess further comprises flowing the first mixture though an anionexchange column to remove excess complexing agent from the mixture. Thefirst reaction product is preferably brought together with a less thanstoichiometric amount of the antibody substance to avoid overlabeling,which could reduce effectiveness or cause side effects.

[0035] Conventional labeling processes of protein based molecules withaugur emitting gamma radioisotopes involves conjugation of thebi-functional agent to the biologic and then this process is followed upwith labeling with a radioisotope. Our alternative method, which iscalled prelabeling, is one in which the bifunctional chelating agent isfirst radiolabeled and then conjugated to the biologic.

[0036] Radiolabeling of antibodies has conventionally been accomplishedby prolonged incubation of the conjugate with a radioisotope solution atroom temperature. This may, however, result in significant radiolysis ofthe protein, such as the antibody structure. Rapid and efficientincorporation of the radioisotope onto the bioactive vector (antibody)is demanded in order to afford a high yield and specific activity of theradiolabeled immunoconjugate.

[0037] The prelabeling procedure of the invention is defined in thefollowing scheme and contains three basic steps:

[0038] 1. Radioactive chelate formation in the absence of an antibody(vector)

[0039] 2. Chelate purification

[0040] 3. Antibody (bioactive vector) conjugation

[0041] With the invention, any combination biologic or bioactivesubstance (vector) may be conjugated with an Auger emitting radionucleotide. An exemplary procedure of prelabeling is as follows:

[0042] Step 1

[0043] A bi-directional chelating agent DOTA-Glys3-L(p-isothiocyanate)-Phe-amide was prepared. Carrier free radioisotope (Yt90 or In 111) in 0.05 M HCL was dried in a heating block under N2 and100 uL of mM 1 in 0.2M ammonium acetate (pH 5.0) was added. This mixturewas incubated at 37 degrees C. for 30 minutes and then 25 uL of 50 mMDTPA in 0.1M ammonium acetate, (pH 6.0), was added for 15 minutes atroom temperature (to complex any remaining radioisotope). The solutionwas loaded into anion-exchange resin column and this column was spun for2 minutes at about 2000 g, and this was followed by elution with four125 uL aliquots of sterile purified water by centrifugation at about2000 g each. Most of the radioactive chelates for step 2 are recoveredin the first four fractions.

[0044] Step 2

[0045] All of the eluted fractions are collected and concentrated toabout 15 uL with a speed-vac concentrator, a step that may be avoidedwhen higher amounts of radioactivity are used. The radiochemical purityof both Yt-90 and In-111 was determined to be greater than 9% by gelfiltration HPLC, cellulose acetate electrophoresis and silica gel TLC.Excess chelating agents, complexes containing divalent nucleotides andDPTA complexes are negatively charged. Thus, the DOTA-peptide complexeswith trivalent nucleotides can be filtered quickly through anappropriately designed anion-exchange column in water to separate themfrom anionic species. Thus the neutral chelate avoids the need for morecomplex processes, in step 2, such as HPLC with mixed organic/aqueoussolvents. Prelabeling deals with the impurity problem by using a largeexcess of chelating agent and then removing this excess, but it does noteliminate trivalent nucleotide complexes from the product.

[0046] Step 3

[0047] In the conjugation step, a high concentration of antibody(bioactive vector) is desired. In this process each molecule of thechelate isothiocyanate will frequently encounter biovector amino groupswith which to react. The concentrated solution is mixed with 1 mg ofchimerical mAb (bioactive vector). The pH is adjusted to 9.5 usingaqueous 2.0 M triethylamine. This mixture is incubated at 37 degrees forone hour and was isolated using a centrifuged gel-filtration column. Atthe chosen conjugation conditions of 1 hour incubation at 37C., pH 9.5the conjugate yield was over 40%, but for radioactivity yields of 100mCi or greater radiolysis will become important. While theisothiocyanate group on the bifunctional chelating agent is potentiallysubject to hydrolysis during the labeling and conjugation steps,controlled experiments have demonstrated a loss of less than 5% of theisothiocyanate.

[0048] This procedure has several advantages over conventionalradiolabeling of antibodies. In step one, the nucleotide chelateformation is easier to control because there is little or no competitionform the nucleotide binding sites on the protein and the chelationconditions are not limited by the need to avoid denaturing the proteinbased antibody. In step 2, excess chelating agent may be removed beforethe radioactive chelate is attached to the protein, thus avoiding theproduction of multiply labeled immunoconjugates, each with unfavorablebiological properties. Finally, in step three, the antibody ischemically modified and radiolabeled in one step, thereby minimizing thechemical manipulation of the antibody and reducing losses of theradiolabeled antibody chelate. This prelabeling approach permits the useof a large excess of bifunctional chelating agent to achieve a highchelation yield quickly in step one, but it requires a rapidpurification method to remove unlabeled reagent in step two.

[0049] Radiolabeling of protein conjugates with radioisotopes issensitive to pH, buffer and temperature effects. The optimum pH for thelabeling (step 3) reaction was different for each protein and may berelated to the isoelectric point of the protein. Radioisotopeincorporation at high specific activity was accomplished in acetate andTris buffers, while the presence of citrate inhibited the labelingreaction. Increasing the temperature of the radiolabeling reaction to37-43 degrees greatly increased the efficiency or radioisotopeincorporation and the kinetic stability of the radioconjugates.

[0050] The chelates that are used for labeling comprise less than 5% ofthe total attached chelates on the Ab (bioactive vector). Inconventional methods of labeling, the excess chelating groups may affectthe biological properties of the antibodies by inducing an immuneresponse and impure radioisotope solutions may require larger amounts ofthe immunoconjugate. With prelabeling, however, a far smaller number ofchelates become attached to the antibody and practically all areradiolabeled while the number of multiply modified antibodies isessentially zeroed. These radiolabeled antibodies are nevertheless fullyimmunoreactive. The efficiency of incorporating a radioisotope into abioactive chelate is directly proportional to the pH, for example theincorporation increased form 69% at pH 5.5 to 81% at pH 7. Theimmunoreactivity of radiolabeled Indium 111 and Yttrium 90 ontoantibodies was found to be 98.6%. Covalent attachment of DOTA to aminesby acylation with the isobutyl formate mixed anhydrides of the chelatingagent has been employed to synthesize a variety of DOTA amines. A simplewater soluble chemical procedure for the conjugation of DOTA toproteins, by ester attachment, is enhanced by elevated temperature(37-43 degrees), optimum pH (7-9) and appropriate buffer solutions whichresult in the rapid labeling of radioimmunoconjugates displaying a highspecific activity.

[0051] The common factor in the use of one of these Radionucleotide1-DOTA or DPTA (chelators)biovectors-antibody complexes described aboveis that it attaches only to the surface of the target cells(s) to bedestroyed and the emission of Auger energy (Indium 11 or Yttrium 90) istransmitted in tissue only for short distances (10 nm-15 nm) andduration (2-3 day half-life), this activating one of the apoptotic celldestruction mechanisms. The process of apoptosis and cell lysis ispivotal in the explication of the role of induction ofantigen/antibody-conjugated-radioisotope triggered cell death using ofnew created radiolabeled monoclonal/phage antibodies (mAbs).

[0052] There are two treatment methods according to the invention. Inthe first method, the antibodies produced in response to the livingpathogens are obtained and replicated. The replicated antibodies areconjugated with a radioisotope which has a half-life of less than 100days to produce a therapeutic composition. The therapeutic compositionis then brought together with the pathogens outside of the patientsbody. The second method is carried out by identifying the livingpathogens causing the infectious disease, selecting a therapeuticcomposition comprising an organic moiety which is chemically selectivefor attachment to the pathogens and which is conjugated to aradioisotope which has a half-life of less than 100 days, depositing thetherapeutic composition on support particles, forming a bed of thesupport particles, passing the patients blood through the bed, andreintroducing the patient's blood to the patient. In this manner thepathogens become attached to the therapeutic composition so that theyreceive a fatal radiation dose.

[0053] The preferred radioisotopes to use are those which emit Augerelectrons, and in both methods the organic moiety is generally anantibody substance, usually an immunoglobulin or immunoglobulinfragment. The antibody substance is usually conjugated to theradioisotope with a complexing agent as hereinbefore described. The doseto be administered to the pathogens will vary depending on many factors,but will generally be in the range of 1 to 1000 millicuries and in anamount which is adequate to render at least some of the pathogensnonviable.

[0054] The invention can be applied to treat disease caused by the HumanImmunodeficiency Virus, HIV, the retrovirus that causes AIDS. The virusis a double stranded RNA virus 100-120 nm diameter and as its basicstructure it has a gag (core protein-p24 and matrix protein-p17 and p7),pol (polymerase/reverse transcriptase-p66/51, p32 and p11) and env(envelope protein) genes. On the surface of the virus are twoglycoproteins called the gp 120 and a trans membrane gp 41. The gp120 isresponsible for binding to the surface of uninfected CD4 cells (Tlymphocytes) by a GP120-CD4 linkage. In fact, the HIV gp120 glycoproteinbinds to CD4 resulting in a conformational change that exposes the V3loop in gp120 and permits the subsequent interaction with a chemokinereceptor CXCR4 on the surface of CD4 T cells or CCR5 surface receptor onmacrophages in order to gain entry into these cells. Therefore, thesechemokine co-receptors are critically involved in the subsequentgp41-mediated fusion and cell internalization. There is widespreadimmune dysfunction and the host CD4 cells are killed as the virusreplicates using the reverse transcriptase as mechanism to usurp the CD4cell's own DNA. The spectrum of immune dysfunction is characterized bydepletion of the CD4 T cells, decrease responses to antigens, mitogens,alloantigens and anti-CD3 antibody, a associated with decreased IL-2production as well as other changes in cytokine production. Finallythere is a loss of specific HIV cytotoxic responses and an increase inunresponsive CD8 T cells, increased beta-2 microglobulin and serumneopterin as well as an increase in autoantibodies and immune complexes.The average half life of the virus and other infected cells in thecirculation is less than two days, wherein millions of virions arereleased from infected cells and similar numbers of new cells areinfected daily. Antibodies to core and surface proteins may be detectedin the serum of infected patients within 2-6 weeks after the initialinfection has occurred. Traditional therapy has been nucleoside analoguereverse transcriptase inhibition and polytherapy with non-nucleosidereverse transcriptase inhibitors and carbocyclic nucleoside analogues.Even with aggressive triple-drug combination anti-retroviral therapy, adecrease in HIV-RNA (viral load) plasma levels may not be sustained, andthis indicates viral therapy failure. HIV resistance to anti-retroviralagents is likely to be a significant factor contributing to treatmentfailure in many individuals. This resistance to drug therapy developsbecause of the error rate of the HIV reverse transcriptase and the highreplicative rate of the HIV which leads invariably to frequent mutationsin the HIV genome. Resistance to most anti-retroviral agents has beendocumented in both in vitro and in vivo. There are mechanisms to testfor resistance mutations (genotype analysis) or resistance phenotype forvirus from any given HIV infected individual and cross-resistance isknown to occur.

[0055] The HIV virus envelope glycoproteins are less than idealimmunogens since the gp120-gp41 are associated and are buried in theinterior of the functional envelope glycoprotein spike outer core. Thenon-covalent nature of the association between gp120 and gp41contributes to the lability of the functional envelope glycoproteintiter. Furthermore, the CD4 binding site is recessed and variableregions, which exhibit glycosylation, flank it. Moreover, variableloops, V2 and V3 mask the chemokine receptor-binding site. However,during the natural HIV infection, disassembled envelope glycoproteinselicit most of their antibodies directed toward these viral envelopecomponents. At that time the interactive regions of gp120 and gp41 areparticularly antigenic. However, because the cognate antibodies cannotbind the assembled, functional envelope glycoprotein complex, thesenatural antibodies do not exhibit an effective neutralizing activity.The efficacy of the humoral immune response in vivo is compromised by atleast two factors: the relative resistance of primary virus isolates toneutralization and the temporal pattern with which neutralizingantibodies are generated. HIV viruses that have been passaged inimmortalized cells lines are typically more sensitive to neutralizationby antibodies than are primary clinical isolates. During natural HIVinfections, disassembled envelope glycoproteins elicit most of theantibodies directed to these viral components. Antibodies to theseenvelope proteins typically can be detected in the sera of HIV infectedindividuals by 2-3 weeks after infection. Later in the course of the HIVinfection, antibodies capable of neutralizing a wide range of HIVisolates appear. Human monoclonal antibodies derived from HIV infectedindividuals have been identified that recognizes the gp120 proteins froma diverse range of HIV isolates. Another fairly conserved gp120neutralizing epitope is recognized by the 2G12 antibody and bind thegp120 epitope on the outer domain. This 2G12 antibody may recognize moreconserved carbohydrate structures that have been formed as a result of aheavy concentration of N-linked glycosylation in the gp120 outer domain.In vivo, the apparent rarity with which the 2G12 antibodies are elicitedattests to the success of the viral strategy of using heavilyglycosylated outer domain surface in immune evasion. The use of phage2G12, gp120 and gp41 antibodies will avoid many of the bioactive vectorproblems.

[0056] By exploiting strategies used by the immune system, phagelibraries can produce antibodies with many clinically applicableimmunochemical specificities. From such libraries, many differentantibodies can be isolated against virtually any antigen. Phageantibodies have a number of advantages over monoclonal antibodiesproduced from hybridomas. First the amino acid sequences of theantibodies are entirely human. Second, in vivo mechanisms that normallyget rid of self-reactive antibodies are avoided, because selectionoccurs mainly in vitro. This makes it possible to produce humanantibodies against human antigens. Third the immunoglobulin V genes arealready cloned and this increases the affinity of the antibodies, changetheir fine specificity, and alters their size or valency. Finally, forthis purpose, genetic engineering may be used to fuse the antibodies tocytolytic proteins that enhance the therapeutic effect of theantibodies. The ability to produce these phage antibodies is relativelyrapid (a few weeks). Therefore, phage antibodies to gp120 and gp41 couldbe chelated to a radioisotope with a half-life of 2-3 days such as 100millicuries of Indium 111 (half-life of 2 days) should be givenintravenously to AIDS patients. The course of the RNA viral load, CD4and CD8 T lymphocytes as well as other markers heralds both theprogression and the regression of disease. This potentially curativeextracorporal treatment procedure should be repeated every 4-6 months,while the response of therapy is scientifically evaluated.

[0057] The patients

[0058] Human subjects who are good candidates for treatment inaccordance with the invention are those who are afflicted withblood-borne pathogens which are debilitating and have been unresponsiveto conventional therapies.

[0059] In the case of HIV infection, the patient should meet thefollowing criteria:

[0060] 1. The patient should be 18 years of age or older and confirmedwith AIDS. The patient should have a positive ELISA and Western Blottest for HIV infection.

[0061] 2. The course of AIDS has been either poorly controlled orunresponsive to adequate anti-HIV therapy as confirmed by clinicalevaluation, an RNA viral load of over 10,000 and a CD4 T cell count ofover 250. Patients who are incapable of generating a cellular responsefollowing a significant reduction of RNA viral load are not consideredto be good candidates.

[0062] 3. The patient has at least 2 or more HIV positive test resultswith a viral RNA load of over 10,000.

[0063] 4. The patients is currently stable (for at least 12 weeks) butexhibits poor or no response to anti-viral therapy (viral loadincreasing and/or CD 3, 4, 8 counts decreasing).

[0064] 5. The patient should have adequate bone-marrow (hematopoietic)function as shown by the following:

[0065] a. Peripheral absolute granulocyte count of 1500/microliter orgreater or a total leukocyte count of 3500/microliter or greater.

[0066] b. Platelet count of 75,000/microliter or greater.

[0067] c. Hemoglobin (Hb) and Hematocrit (Ht) at least 60% of normal

[0068] 6. The patient should have adequate hepatic function as shown bythe following:

[0069] a. Serum bilirubin of 2.0 mg/dl or less

[0070] b. Values for SGOT(glutamate oxalacetate transarinase),SGPT(serum glutamate pyruvate transaminase) and alkaline phosphatase ofnot more than three time the normal range.

[0071] 7. The patient should have adequate renal function as shown by aserum creatinine of 2.0 mg/dl or less.

[0072] 8. The patient should not be impaired to the point of beingdisabled, requiring special care and assistance, having severe signs andsymptoms, and having abnormal Laboratory.

[0073] HIV positive (HART Failure-AIDS) patients are preferred.

[0074] The patient is not a good candidate is if any of the followingapply.

[0075] 1. The age of the patient is less than 18 years.

[0076] 2. The patient has a hemoglobin value of less than 8 gm/dl

[0077] 3. The patient has clinically significant abnormalities inglucose, sodium, potassium, chloride, calcium, phosphorus, uric acid, orBUN.

[0078] 4. The patient as Participated in an investigational clinicaltrial or treatment within the previous 30 days;

[0079] 5. The patient has other medical condition in which the study maypose a threat to the patient ps Preparation of The Pharmaceutical

[0080] Radiometal and Antibodies

[0081] In accordance with the invention, a radiometal is conjugated withan antibody for the target blood borne pathogen.

[0082] The Radiometal

[0083] For AIDS treatment, Iodine 131, which is suitably employed inthis application, can be obtained from M.D.S. Nordion with a certifiablepurity of 99.2% and impurities of 0.2%. The batch, validity number anddate are recorded. Following receipt the product is analyzed forimpurities by atomic emission spectra and any impurities are removed bygel filtration with an appropriate chelating agent and final product isthen tested for purity.

[0084] The Antibody

[0085] For AIDS treatment, suitable antibodies can be obtained fromResearch Diagnostics, Inc. HIV gp 120, a recombinant human cell derivedantibody having a molecular weight of 120,000 Daltons is lyophilized andis of isotype IgG1 produced in Baculovirus system and binds to HIV-1 gp120 envelope antigen on the V 3 loop, but it does not neutralize theHIV-1 MN, HIV-1SP2, HIV-IIIRF or the HIV-1SF2.

[0086] Forming the Antibody-radiometal Conjugate

[0087] Iodine 131 binds directly to the tyrosine aromatic ring 1,5positions of the gp120 antibody in-vitro. Therefore, recombinant gp120-antibodies are labeled with Iodine 131 by direct incubation chemicalreaction. The following mixture I 131-gp120 antibody is tested for boundand unbound I 131 and the latter (unbound 1 131) is eluted from thegp120-I 131.

[0088] Adherence of the Conjugate to a Suitable Support

[0089] After I 131 and gp120 binding the radioactive compound is thenreacted with Protein A or Protein G coated 100-500 mm beads and theexcess I 131-gp120 antibody-compound is washed and passed over ananion-bound resin for removal of traces of antibody-I 131 compound. Theresultant beads bind the antibody-I 131 compound in their Fc portion,which correctly orients these antibody complexes for maximum antigen(gp120/antigen on the HIV viral plasma membrane surface) trapping andcapture. Protein G captures more antibody subclasses than the Protein Aand is therefore preferred for this application.

[0090] Stability Testing of the Conjugate

[0091] A test is performed to verify the stability of the conjugateunder the conditions within the canister by taking an aliquot (10-50microliters) of the radiolabeled conjugate containing approximately8×10(6) cpm of I 131 radioactivity and adding the conjugate to 1.1milliliters of fresh human serum containing 10 microliters of 10% NaN₃The mixture was incubated at 37 degrees Centigrade throughout the study,during which 100-microliter samples of serum were analyzed by gelfiltration BPLC on two Pharmacia Superose 12 HR 10/30 columns (1×30 cm)in series, using an isocratic mobile phase of 0.05 M Na₂SO₄, 0.02 MNaH₂PO₄, 0.05% NaN₂, at pH 6.8. Serum samples are analyzed at intervalsranging from 15 to 72 hours for 10 days to determine conjugatestability.

[0092] Preparation of a treatment bed containing the conjugate

[0093] The beads are placed in a canister that allows whole blood tofreely pass through the pores in the canister but without allowing thebeads to escape. Generally speaking, the bed contains on the order of100 mCi of I-131 or other radioisotope bound to approximately 10micrograms of antibody, via tyrosine residues, for example. Theconjugate is attached to the beads via a protein, for example, whichbinds to the substrate to the antibody. Functionally speaking, the bedpreferably contains adequate conjugate to bind 98% or more of the bloodborne pathogen in the patient's blood in three passes of the patient'sblood volume through the canister. This is preferably accomplished in aperiod of a few hours.

[0094] Use of the Treatment Bed

[0095] Whole heparinized-blood is used into the extra-corporal device. Adouble lumen IV catheter is inserted in the FHV positive patients.Peripheral blood will be analyzed before and after the wholeblood-pheresis. The catheter is connected to the device and the flowrate is adjusted to 500-1000 milliliters per hour or about 2-4 drops persecond. This rate allows for complete exchange to occur in 8 to 10 hoursand during the 7-8 day period of whole blood exchange, a passage of 21times through the canister. Continuous monitoring of the radiationexposure will be made during the procedure. Each patient will be placedon the whole blood I 131-gp120 antibody dialysis for 8 days in outpatient therapy (renal dialysis) unit. Renal dialysis may be performedfor a period of 30 or more days if necessary, without untoward effects.

[0096] Theory

[0097] Specific and unique preceptors (located on the surface of allliving cells) are on targeted celled as unique membrane antigens(usually glycoproteins that extend from the cell membrane surface) thatare employed for the targeted cell to attach to normal cells. They arecalled intgrins, selectins and adhesion molecules. Recombinantlyophilized monoclonal antibody is obtained against these specific andunique antigens. A radiometal of suitable half-life and intensity isselected for emitting energy during the life cycle of the targeted cell.A radionucleotide-monoclonal antibody is formed from these componentsfor use in treating the blood of the patient.

[0098] In the case of HIV/AIDS it is known that the AIDS virus has alife cycle of 48 hours in humans and there is neither a known cure noran adequate animal models for simulation of the human HIV disease. Whenthe radiometal (Iodine 131) is bound to the HIV cell surface antibody(gp120), which in turn, attaches to the HIV cell surface antigen, theradiometal will destroy the gp 120 antigen and the virus itself.Lymphocyte adaptive response is likely to occur following therapy ordestruction of the HIV virus.

[0099] The Gp 120 viral antigen normally is hidden but prior toattachment and immediately after exit (virions) it is exposed. The V3loop is the active attachment to the CD4 marker on the host Tlymphocyte. The GP 120 monoclonal antibody has 22 tyrosine linkages onthe V1, V2, V4, and V5 loops, but none on the V3 loop. The binding ofbinding of I 131 occurs chemically with the tyrosine linkages. Thebinding to tyrosine is firm and not easily capable of dissociation. Forexample, I 131-tyrosine binding is used in thyroid irradiation.

[0100] The recent development of an efficient process of biologicalchelating allows for the adherence of antibodies to a radiometal, andmakes it possible to produce compounds that are both targeted becausethe antibody is specific for only one antigen on the cell surface anddelivers the potential destructive fore (Iodine 131 gamma emission) inwater and tissue.

[0101] For application of the invention, the measurement of the I 131isotope in free form is easily performed. The measurement of the effectof the treatment on the patient is also readily performed.

[0102] Technology for extra-corporal therapy is standard medicalpractice in the dialysis and plasmapheresis fields. Technology existsfor antibody (gp 120) capture (on beads or columns) is available usingProtein A or Protein C coating.

[0103] The gp 120 antibody/I 131 binding to the THV viral gp 120 antigen(receptor) follow the accepted formula for peptide-receptor binding.Generally speaking, the affinity for a peptide with its receptor may beseen in the following formula:

[0104] (PR)/P+R=K×M

[0105] where P is the circulating peptide, in this case gp 120-I 131

[0106] R is the peptide specific receptor, in this case Viral gp 120receptor,

[0107] PR designates the peptide bound to the specific receptor,

[0108] K is the affinity constant, and

[0109] M is the weight in grams of a single peptide molecule.

[0110] If r is express as the micrograms of gp 120-I 131 bound to viralprotein gp 120 antigen M.

[0111] As there are independent binding sites on each virus (gp-120antigens) these may be assigned as v. Therefore,

r=v(K−gp120-I131)/1+(K−gp120-I131).

[0112] since the binding sites on the viral gp 120 are all of the sameclass and chemical structure a and molecular configuration.

[0113] The mathematical formula may be derived from a standard Langmuirplot as:

r=Sigma(n−1)VK(gp120-I131)/1+(K(gp120-I131)+vηKη(gp120-I131)

[0114] For gp 120 antibody-viral gp120 antigen, this affinity may bemeasured and is very high, on the order of 1.9933 (10²⁰).

[0115] Specific Methods

[0116] The recombinant HIV-1 glycoprotein gp 120 antibody is bound toIodine 131 (half life 8.02 days) in the following concentrations:

[0117] a) 0.5 mCiI 131 MDS Nordion

[0118] b) 1.0 mCiI 131

[0119] c) 2.0 mCiI 131

[0120] d) 5.0 mCiI 131

[0121] The method of measurement for effectiveness of therapy is amplydiscussed herein.

[0122] The measurement of radioactivity of pre and post therapy linesand peripheral blood of the patient may be immediately determined and,if necessary, the therapy may be discontinued.

[0123] A dose response curve may be evaluated and repeat therapy may beperformed as needed.

[0124] The following schedule will be maintained following consent hasbeen obtained.

[0125] Each patient will have a double lumen intravenous catheter placedin the appropriate and initial sample of blood drawn for the initialbaseline CD3, CD4, CD8, CD38, RNA viral load, DNA flow studies.Following these initial studies, each extra-corporal system of slowinfusion over the I 131 0.5-5 m Ci-gp120 antibody-beads over a period of192 hours (8 days).

[0126] On the basis of these methods, the most appropriate dose ofIodine 131-gp120 monoclonal antibody beads can be produced withverifiable quality control.

[0127] Because of the variables for prognostication for AIDS therapy,which includes rapid mutation of the HIV virus, the host response andthe host reservoirs (dendritic cells, macrophages and semen) it isprobable that, if successful, additional extra-corporal therapy may benecessary. During the course of follow-up evaluations, it may benecessary to repeat the therapy with the objective of reducing the RNAviral load to undetectable levels and with gradual individualimprovement of CD3, CD4, CD8 counts to normal levels.

[0128] The patient will return to Out Patient Suite for insertion of thedouble lumen catheter and for slow extra-corporal venous infusion ofheparinized whole blood over extra-corporal columns of isolatedradioactive beads of Iodine 131-gp120/41 antibodies over 192 hours (8days).

What is claimed is:
 1. A composition of matter comprising a conjugate ofa living pathogen-targeting organic moiety coupled to a radioisotopewhich has a half-life of less than 100 days, said conjugate beingdeposited on a support.
 2. A composition as in claim 1 wherein thesupport is in the form of beads.
 3. A composition as in claim 2 whereinthe conjugate is bound to the support.
 4. A composition of matter as inclaim 3 wherein the living pathogen-targeting organic moiety targets avirus.
 5. A composition of matter as in claim 4 wherein the livingpathogen-targeting organic moiety comprises a human immunoglobulin or ahuman inmmunoglobulin fragment.
 6. A composition of matter as in claim 1wherein the radioisotope emits Auger electrons.
 7. A composition ofmatter as in claim 6 wherein the radioisotope has a half-life in therange of from about 1 to about 10 days.
 8. A composition of matter as inclaim 7 wherein the radioisotope is selected from the group consistingof Phosphorus 32, Copper 67, Gallium 67, Bromine 77, Yttrium 90,Technetium 99, Indium 111, Iodine 125, Iodine 131, Rhenium 186, Rhenium188, Platinum 195, Bismuth 213, and Astatine
 225. 9. A composition ofmatter as in claim 8 wherein the radioisotope consists essentially ofIodine
 131. 10. A method for treating an infectious disease caused byliving blood-borne pathogens in a mammal, wherein said mammal producesantibodies in response to said living pathogens, said method comprisingobtaining antibodies from said mammal; replicating said antibodies toproduce replicated antibodies, conjugating said replicated antibodieswith a radioisotope which has a half-life of less than 100 days toproduce a conjugate, fixing said conjugate to a conjugate support toform a supported conjugate, and passing the blood of said mammal incontact with said supported conjugate to bring said conjugate intocontact with said living pathogens.
 11. A method as in claim 10 whereinsaid supported conjugate is in the form of particles, said methodfurther comprising forming a bed of said particles, flowing the blood ofsaid mammal through said bed to form treated blood, and returning thetreated blood to said mammal.
 12. A method as in claim 11 wherein themammal is a human and the pathogen is HIV virus.
 13. A method as inclaim 12 wherein the antibody is selected from the group consisting ofgp120 antibody and gp2G12 antibody and the radioisotope is Iodine 131.14. A method for treating an infectious disease caused by livingblood-borne pathogens in a mammal, said method comprising identifyingthe blood-borne pathogens causing the infectious disease, selecting asupported conjugate comprising a particle support bearing an organicmoiety which is chemically selective for attachment to said livingpathogens and which is conjugated to a radioisotope which has ahalf-life of less than 100 days, flowing the blood of said mammalthrough a bed formed from particles of said supported conjugate, so thatsaid blood-borne pathogens become associated with said radioisotopewhile in the bed, forming treated blood, and returning the treated bloodto said mammal.
 15. A method as in claim 14 wherein the organic moietyis selected from the group consisting of an immunoglobulin and animmunoglobulin fragment.
 16. A method as in claim 15 wherein theradioisotope emits Auger electrons.
 17. A method as in claim 14 whereinat least some of the living pathogens are rendered non-viable while inthe bed.
 18. A method as in claim 17 wherein the living pathogen is avirus.